Created: Sep 2021
Enzymes are proteins folded into complicated spatial structures. They facilitate various reactions which are crucial for sustaining life functions of organisms. Enzymes act as catalysts, making reaction speed millions of times faster.
All commercially available enzymes are produced by organisms, especially microorganisms. Among them, filamentous fungi can secrete plenty of enzymes to degrade extracellular material and obtain nutrient for their growth. Therefore, filamentous fungi have been used as an enzyme producer. In this experiment, we investigate the enzyme activity of cellulase (cellulose degrading enzyme) derived from filamentous fungus Trichoderma reesei.
When T. reesei is cultivated on the medium containing cellulose as the sole carbon source, it produces a large amount of cellulase. However, T. reesei does not produce cellulases when easily metabolizable carbon sources are used. In this experiment, we will use culture fluids derived from cultivation on cellulose and glucose.
The purpose of this experiment is to find the value of the activity of cellulase using the T. reesei cultivated on cellulose. Moreover, we will investigate whether the T. reesei cultivated on glucose can also break down CMC cellulose (whether it contains cellulase).
a. 500 mM acetic acid buffer (pH 5.0)
b. 50 mM acetic acid buffer (pH 5.0)
c. 1.25 % carboxymethylcellulose (CMC
d. 3,5-Dinitrosalicylic acid (DNS) reagent
e. 0.5 M sodium hydroxide.
f. 10 mg/mL glucose
2.1. Preparation of standard curve (Day 1)
Cellulase activity is determined by its effect on cellulose with respect to reducing sugar formation. Reducing sugar can be detected by DNS method. In this experiment, glucose is used as the standard material of reducing sugar.
In DNS method, when 3,5-dinitrosalicylic acid (DNS) and reducing sugar (glucose) are reacted under strongly alkaline conditions, the yellow DNS is reduced to form red 3-amino-5-nitrosalicylic acid. Since the increase in absorbance at 500 to 540 nm in this reaction is proportional to the concentration of the reducing sugar, quantification of reducing sugar can be made from a standard curve obtained using a reducing sugar solution of a series of known concentration.
To determine the amount of reducing sugar, we plot a graph of absorbance for different concentrations of glucose solution (standard curve).
1) Using the table below as reference, prepare 0, 1, 2, 3 mg/mL glucose solutions (10mL of each).
2) Pipet 200 uL aliquots into 3 test tubes per each concentration (12 test tubes in total). Add 400 μL of DNS reagent, mix well. Boil for 5 minutes. Add 1800 μL of distilled water, mix well.
3) Using the distilled water as blank solution, measure the absorbance of each sample at 540 nm wavelength with the spectrometer. To obtain the absorbance values to plot on the graph, deduct the average value of absorbance for 0 mg/mL glucose concentration from the absorbance values for all the other concentrations.
4) Make a graph (x axis: glucose concentration, y axis: absorbance) and plot 3 points for each concentration. Calculate the gradient (inclination) of the graph.
2.2. Enzyme assay (Day 2)
For the measurement of enzyme activity, it is necessary to strictly control the composition, pH, temperature and reaction time.
The unit of enzyme activity is U (=µmol/min). We find the enzyme activity by measuring the decrease of the substrate amount/ increase of product amount per given time unit. From the experimental results, we find the enzyme activity per 1mL of cellulase solution.
When the concentration of the substrate if high enough, initial speed of the reaction and enzyme activity amount are proportional. In other words, the graph of reaction time (x axis) vs. product amount (y axis) becomes a straight line.
1) Pipet 200 µL aliquots of culture fluid of T. reesei from cellulose cultivation into 18 test tubes. Heat 15 test tubes in 50°C bath. Add 800µL of CMC solution (at 50°C) to 15 of the test tubes at times designated in Table 2 and mix. Maintain the temperature of the test tubes at 50°C. Per every 3 test tubes, incubate the solution for 2, 4, 6, 8, 10 min. At times designated in Table 2, add 100 µL of 0.5 M NaOH to the respective test tubes and mix the solution.
In case of the remaining 3 test tubes, there is no need to control the temperature. Add 800µL of CMC, mix, add 100 µL of 0.5 M NaOH and mix again. Those solutions will be used as blank solutions (reaction time= 0 min).
2) Place 200 µL of incubated solutions from 18 test tubes into clean test tubes. Add 400 μL of DNS reagent, mix well. Boil for 5 minutes. Add 1800 μL of distilled water, mix well.
3) Measure the absorbance of the 18 solutions at 540nm. Use distilled water as the blank solution.
Next, calculate the average of 3 samples of reaction time= 0 min. To obtain the absorbance values to plot on the graph, deduct the average value of absorbance for 0 min incubation time from the absorbance values for all the other reaction times.
4) Make a graph (x axis: reaction time, y axis: absorbance) and plot 3 points for each concentration. Calculate the enzyme activity of cellulase using the absorbance value at reaction time = 10min and the standard curve from 2.1.
5) To investigate whether the T. reesei cultivated on glucose can also break down CMC, carry out 10 min reaction (incubation) for solution of culture fluid of T. reesei from glucose cultivation, following the method in 1)-3). Prepare 3 test tubes of solution for 10 min reaction, and 3 test tubes of blank solution (reaction time = 0 min). Compare the results when glucose cultivation is used to results when cellulose cultivation is used.
3.1 Preparation of standard curve (Day 1)
The Absorbance Adjusted value is equal to Absorbance Raw – average absorbance at 0 mg/mL glucose concentration.
For the 3 mg/mL glucose concentration solution, the precision of the 3 measurements was very low, with one of the solutions having an anomalous absorbance of 1.000. That value was not taken into consideration when plotting the best fit line.
To make up for lack of precision of the first few measurements, the experiment was repeated for 3 mg/mL glucose concentration solution. The precisions of those 3 measurements was acceptable, but they were to high to be on the best fit line with the data for the other 2 concentrations of glucose. Nevertheless, the standard curve in Figure 1 was plotted using the data from Table 3.
The possible reasons for low accuracy and precision of the absorbance data for the 3 mg/mL glucose concentration solution will be listed in the Discussion section.
3.2 Enzyme assay (Day 2)
Absorbance for blank solution (reaction time = 0 min) was 0.019 for all 3 samples, thus the spectrometer was set to Auto Zero so that the Absorbance Raw = Absorbance Adjusted (no need to deduct average absorbance at 0 min from absorbance at other reaction times). Results are displayed in the Table 4. The values from Table 4 were used to plot the graph in Figure 2.
3.3. Activity calculation
Based on the data from Figure 2, we know that the absorbance value for 10 min reaction time is 0.246. The standard curve in Figure 1 is defined by the y= 0.48x formula, so the glucose concentration corresponding to 0.246 absorbance is as follows.
Glucose concentration = x = y ÷ 0.48 [mg/mL] = 0.246 ÷ 0.48 [mg/mL] = 0.5125 [mg/mL]
Moles of glucose produced = (glucose concentration × 1.1 × 1000) ÷ 180 [µmol]
Glucose produced per minute = Moles of glucose produced ÷ 10 [U]
Cellulase activity per 1 mL enzyme solution = Glucose produced per minute / 0.2 [U/mL]
Combining the 3 lines above into one formula:
Cellulase activity per 1 mL enzyme solution = (glucose concentration × 1.1 × 1000) ÷ (180 × 10 × 0.2) [U/mL] = (0.5125 × 1.1 × 1000) ÷ (180 × 10 × 0.2) [U/mL] = 1.566 [U/mL] ≈ 1.6 [U/mL]
In addition, 10 min reaction (incubation) for solution of culture fluid of T. reesei from glucose cultivation was carried out. The results are displayed in Table 5. The significance of the results for the glucose cultivation, and comparison with the results for the cellulose cultivation will be carried out in the Discussion section.
On day 1 we created the standard curve in Figure 1. For glucose concentrations 1 mg/mL and 2 mg/mL, the points on the graph are quite close together, indicating adequate precision of the measurements. However, in case of data points for glucose concentration 3 mg/mL, the precision is very low, not a single point being on the line of best fit. The 1.000 absorbance point is especially anomalous, and probably occurred because of adding 2 mg/mL solution into the test tube, instead of 3 mg/mL solution.
The data for the first 3 samples of 3 mg/mL glucose is below the line of best fit, and the data for the 3 samples from the redo of the experiment are all above the line of best fit. The precision of the last 3 measurements is satisfactory, but the absorbance is too high to fit with the results from other concentrations, which hints towards a systematic error in the measurements. It shows that the last 3 solutions for 3 mg/mL glucose were not dilute enough compared to the 1 mg/mL and 2 mg/mL glucose.
In Figure 2, the precision is much better, although it is far from ideal for 6 min and 10 min reaction times. This is probably mostly due to human error- the times of addition of CMC and NaOH have probably slightly differed to the designated times in Table 2, giving rise to a decrease in precision and accuracy.
More importantly, the graph of time vs. absorbance, contrary to expectations, did not become a straight line, but a curve. When the concentration of the substrate if high enough, initial speed of the reaction and enzyme activity amount are proportional, so a straight line is expected. The concentration of cellulose in the culture fluid of T. reesei cultivated on cellulose was high enough, so this is not a possible reason why the graph became a curve. Temperature in the water bath was constant, and when room temperature NaOH was added at the end of the reaction, the test tube was removed from the water bath, so it is also not the reason for the curve to form. However, as the CMC breakdown reaction progressed, more and more glucose was produced. As the number of glucose molecules in the solution increased, it has become slightly rarer/slower for a cellulase molecule to find a CMC molecule to react with and break it down. Therefore, as the time of the reaction and the concentration of glucose in the solution increased, glucose became an inhibitor for the CMC breakdown reaction and decreased the amount of newly produced glucose, thus decreasing the absorbance values and causing the graph in Figure 2 to become a curve.
Using data from Figure 1 and Figure 2, we found the concentration of glucose in the solution at 10 min reaction time. Using this value, we calculated the activity of cellulase per 1mL of enzyme (1.6 U/mL). This result means that we need 1.6mL of cellulase to break down 1µmol of CMC (cellulose) in 1 minute.
In case of the solution of culture fluid from glucose cultivation, the absorbance after 10 min reaction time was close to, but not equal to 0. Nevertheless, the absorbance was very low compared to the solutions of culture fluid from cellulose cultivation. Low absorbance at 540 nm reflects low concentration of 3-amino-5-nitrosalicylic acid, and thus low concentration of the reducing sugar- glucose. Glucose is the product of CMC breakdown, so low absorbance of the sample means that only a very small amount of CMC was broken down. This is due to low concentration of cellulase in the culture fluid. The culture fluid was cultivated on glucose, which is an easily metabolizable carbon source compared to cellulase. Thus, having a ready supply of glucose, T. reesei from glucose culture did not need to produce cellulase to break down cellulose. As the result, when culture fluid from glucose cultivation was mixed with CMC, it was not capable of breaking it down into glucose, as it did not contain much cellulose.
T. reesei from glucose sample aren’t expected to produce any cellulase, so the absorbance value of 0 is expected. However, absorbance was higher than zero, which may indicate that even in presence of glucose, a small amount of cellulase is still being produced. Alternatively, the samples may have been contaminated with the cellulose culture fluid.
By investigating the absorbance of glucose, the experimental value of the activity of cellulase produced by T. reesei cultivated on cellulose was established as 1.6 U/mL. In addition, based on the experimental results, it can be deduced that culture fluid solution of T. reesei cultivated on glucose can break down only a very small amount CMC (cellulose) compared to the culture fluid solution T. reesei cultivated on cellulose. Thus, there is a high probability that T. reesei does not produce a significant amount of cellulase when preferable carbon sources are available, which demonstrates the dependence of enzyme production of T. reesei on the carbon source.
By investigating both cellulose and glucose cultivation and determining the cellulase activity, the aim of this experiment was fulfilled.
Dependence of enzyme production of filamentous fungi on the carbon source
As explored in this experiment and explained above, the type of enzyme produced by filamentous fungi is strongly dependent on the carbon source available. By producing the specific enzyme that allows for the carbon source available in the fungi’s environment to be broken down and used as nutrition/ energy source, fungi adapt to various environments and thus their survival chance increases.
However, production of enzymes uses up fungi’s energy and resources, so it is desired to produce only the enzymes that can be used to break down the carbon sources currently available in fungi’s surroundings. Thus, as we saw in this experiment, T. reesei produces cellulase only when cellulose is present in the solution. When an easily metabolizable energy source like glucose is available, T. reesei does not use up its resources to produce cellulase.
Apart from T. reesei, another organism whose enzyme production depends on the species of yeast called Saccharomyces cerevisiae, which uses glucose as a preferential carbon source, but it can also use other sources such as galactose and maltose.
Relationship between enzyme activity and pH/temperature
One of the many factors affecting the activity of an enzyme is the pH of the solution. The reason why pH has an effect on enzymes is because it can change their ionisation state, altering their structure and thus the ability to bind to substrates and facilitating reactions. Change of the ionisation state in enzymes occurs because of the fact that enzymes, which are proteins (α-amino-acid compounds), contain carboxyl and amine functional groups, which can be ionised depending on the pH of the solution.
Another factors affecting enzyme activity is temperature. For example, in higher temperature, reaction speed increases due to more frequent and higher energy collisions of the molecules, but the bonds inside of the protein become weaker, and the protein denaturises. The temperature at which rate of reaction is highest without the protein denaturising (highest enzyme activity) is called the optimum temperature.
Methods of determining reducing sugar
Reducing sugar is a sugar that contains (or is capable of forming) an aldehyde or ketone, and thus is able to act as a reducing agent in a reaction. Apart from glucose, lactose, fructose and maltose are also considered to be reducing sugars.
One way to determine reducing sugar (glucose) is to run a Benedict’s Test using Benedict's reagent. Benedict's reagent contains anhydrous sodium carbonate, sodium citrate and copper (II) sulphate pentahydrate. Reducing sugars are able to reduce the blue copper sulphate to red brown copper sulphide precipitate.
The experimental method is as follows: first, a sample of sugar (or food etc.) is dissolved in boiling water, and several drops of Benedict's reagent are added to the solution. The solution is left to cool, and after several minutes it begins to change colour. In case of blue color, we can deduce that no glucose is present. If glucose is in fact present, depending on its concentration, the solution will be green-yellow (low concentration) to red-brown (high concentration)
Another way to test for reducing sugars (monosaccharides, especially aldoses and ketoses) is Fehling's Test using Fehling's solution. Fehling's solution is made up of 2 sub- solutions: copper(II) sulphate pentahydrate dissolved in water and potassium sodium tartrate tetrahydrate + NaOH dissolved in water. The same amount of both sub- solutions is added to the tested sample. If a reducing sugar is present, aldehyde oxidises to acid, cuprous oxide being formed as a result. Due to encountering an aldehyde group, it is then reduced and forms a red precipitate cuprous ion.
The experimental method is as follows: a solution of the tested sample is diluted by adding water, then it is heated until the sample dissolves completely. In the next step, we add Fehling's solution to the sample solution while mixing it. If the sample solution doesn’t contain any reducing sugar, the color will be green-blue. However, if the sample contains reducing sugars, we will observe red precipitate.
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